It is required to detect a target in various fields such as clinical medical care, food, and environment. In the detection of a target, interactions with the target are generally utilized, and among them, a method using an antibody that specifically binds to a target is widely used. In this method, for example, a target is bound to an antibody labeled with oxidoreductase such as peroxidase. Then, a chromogenic reaction is conducted by the enzyme in the labeled antibody using a chromogenic substrate, and the development in color is detected. By the detection of the development in color, analyses of the target such as qualitative analysis and quantitative analysis are performed indirectly.
However, since the antibody is obtained by immunizing animals, it is really difficult to obtain an antigen specific to a toxic target or a low-molecular-weight target. Hence, recently, a nucleic acid that binds to a target, i.e., a nucleic acid aptamer (hereinafter, merely referred to as an aptamer) has been focused on. The aptamer can be obtained in a test tube. Therefore, for example, it is possible to obtain aptamers to a toxic target and a low-molecular-weight target. Further, it has been attempted to use such aptamer as substitute for the antibody in detection of a target in combination with DNAzyme exerting catalytic activity as in peroxidase. The DNAzyme generally is DNA that exerts a catalytic property of peroxidase by having a guanine-rich structural motif, having a G-quadruplex structure, and forming a complex by binding to hemin.
In the detection of a target, a single-stranded nucleic acid element obtained by linking a single-stranded aptamer and a single-stranded DNAzyme is specifically utilized (Non-Patent Document 1). The single-stranded nucleic acid element forms a stem structure by self-annealing in the absence of a target, and the DNAzyme cannot form G-quadruplex by the stem structure. Therefore, the DNAzyme in the single-stranded nucleic acid element cannot bind to hemin and cannot achieve a catalytic property in the absence of a target. On the other hand, in the presence of a target, the single-stranded nucleic acid element releases the stem structure by binding the target to the aptamer. Therefore, in the presence of a target, the DNAzyme in the single-stranded nucleic acid element forms G-quadruplex and exerts the catalytic property by binding to hemin. Thus, when a chromogenic substrate to redox activity is present together, a chromogenic reaction occurs in the presence of the target, and a chromogenic reaction does not occur in the absence of the target. Therefore, it becomes possible to analyze the target by detecting the chromogenic reaction. Furthermore, it is not required to label the target, and thus, it becomes possible to directly detect various targets including a low-molecular-weight target.
As described above, the single-stranded nucleic acid element is required to control activity of the DNAzyme by a conformation of the aptamer. Therefore, for example, it is desired to combine DNAzyme whose activity can be easily controlled according to the sequence of an aptamer to be used.
However, there is only a limited number of DNAzymes that have been reported. Therefore, when a combination with a predetermined aptamer is decided, there has no choice but to select from the limited number of DNAzymes. Thus, there is a limitation in structuring a nucleic acid element with superior accuracy according to the target. Moreover, in order to make it possible to perform detection with superior sensitivity, DNAzyme which highly exerts redox activity is required.
Therefore, it has been attempted to obtain novel DNAzyme. However, in screening of DNAzyme, there has no choice but to determine each of activities of candidate nucleic acid molecules, and the operation thereof is really complicated.